Methods for the detection of nucleic acids are known from the art. The patent specification U.S. Pat. No. 6,812,334 B1 discloses a method for the detection of nucleic acids. This method comprises the provision of nanoparticles, to which oligonucleotides are attached and of one or several kinds of connecting oligonucleotides. Each connecting oligonucleotide has two sections. The sequence of one section is complementary to the sequence of one of the sections of the nucleic acid and the sequence of the other section is complementary to the sequence of the oligonucleotides on the nanoparticles. The nanoparticle oligonucleotide conjugates, the connecting oligonucleotides and the nucleic acid are combined under hybridisation conditions and a detectable change occurs. The said patent specification also discloses a kit for carrying out the said method.
The European patent application EP 1 870 478 A1 discloses a biosensor, which consists of metal particles, which are fixed to the surface of a carrier. A probe molecule is fixed to these metal particles, wherein the probe molecule, in particular, may be a nucleic acid, which forms a hairpin structure. The probe molecule shows a fluorescence molecule. Before the reaction of the probe molecules with the target molecules, the distance between the metal particle and the fluorescence molecule is equal or less than 5 nm, such that the excitation energy of the fluorescence molecule is transferred to the metal particle. Thereby, the fluorescence quenched. The distance between fluorescence molecule and metal particle is greater after the reaction, such that the fluorescence molecule can fluoresce.
The patent application US 2010 0075335 A1 discloses a colorimetric method for the detection of specific nucleic acid sequences, inclusive of mutations and single nucleotide polymorphisms (SNPs) in nucleic acid sequences by aggregation of nanoparticles. In this method, identical nanoprobes with oligonucleotides are used, which are directly attached to gold nanoparticles, which appear red in a solution. The aggregation of these nanoprobes by an increase of ionic strength leads to a colour change from red to blue. The presence of the target DNA with a sequence, which is entirely complementary to the sequence of the nanoprobe prevents this aggregation and the solution remains red. The method is highly sensitive and makes it possible to differentiate sequences entirely complementary from sequences with one SNP. This patent application also discloses a kit for the application of the said method.
In the publication “Molecular beacons: probes that fluoresce upon hybridization” in Nature Biotechnology, 1996, volume 14, pages 303 to 308, S. Tyagi et al. report a novel principle for the production of probes, which are useful for the detection of specific nucleic acids in homogeneous solutions. Probes on the basis of this principle are single stranded nucleic acids molecules, which possess a stem loop structure. The loop part of the molecule is a probe sequence, which is complementary to a predetermined sequence of a target nucleic acid. The stem is formed by the hybridisation of two complementary arm sequences, which lie on either side of the probe sequence. The arm sequences are independent of the target sequence. A fluorescence donor group is added to one end of one arm and a non-fluorescing, quenching fluorescence acceptor group is added to the end of the other arm. The stem holds these two groups in great proximity, thereby the fluorescence of the fluorescence donor group is quenched by fluorescence resonance energy transfer. When the probe meets a target molecule, it forms a hybrid, which is longer and more stable than the hybrid formed by the arm sequences. In this way, the probe experiences a conformation change, which moves the arm sequences apart. As the fluorescence donor is not close to the fluorescence acceptor anymore, fluorescence is produced. These probes are described as molecular beacons.
Q. Luan, et al. present an E. coli DNA ligase biosensor in their publication “Hairpin DNA probe based surface plasmon resonance biosensor used for the activity assay of E. coli DNA ligase” in Analyst, 2010, volume 135, pages 414 to 418. In this sensor, a hairpin DNA probe is fixed to a gold film and hybridised to two single stranded DNA segments and, in this way, forms a hybrid with a discontinuity. When E. coli DNA ligase is present, the discontinuity is ligated, which leads to a conformation change of the hairpin DNA from the stem loop structure to a stiff double helix. This conformation change results in a change in the surface plasmon resonance.
Y. He et al. in their publication “‘Visual detection of single nucleotide polymorphism with hairpin oligonucleotide functionalised gold nanoparticles” in Analytical Chemistry, 2010, volume 82, pages 7169 to 77 describe a lateral flow strip biosensor, in which a hairpin oligonucleotide is conjugated on its 5′-end with a gold nanoparticle and on its 3′-end it is modified with biotin. The hairpin oligonucleotide on the nanoparticle surface holds the biotin groups close to this surface, which leads to the biotin becoming “inactive”. In this publication, the SNP detection is based on the unique properties of a hairpin oligonucleotide for the molecular differentiation of entirely complementary DNA from DNA with a single base change and the resulting different number of “active” biotin groups on the surface of the gold nanoparticles. After the hybridisation reaction, the gold nanoparticles, which contain activated biotin, are caught in the test zone of the lateral flow strip biosensor provided with immobilised streptavidin. The accumulation of gold nanoparticles in the test zone leads to a characteristic red band, whereby SNPs can be detected visually.